1. Field of the Invention
The invention relates to ciliary neurotrophic factor (CNTF) variants which, owing to the loss of affinity for the interleukin 6 receptor (IL-6R), can be used as a therapeutic with particularly low side effects for disorders or diseases which are associated with CNTF.
2. Description of Related Art
During the development of new medicaments which not only reduce weight safely and effectively, but at the same time positively influence metabolic disarrangements which are often associated with adiposity, such as insulin resistance and glucose intolerance, a class of molecules naturally produced by the body has become the subject of current investigation. These molecules are the so-called glycoprotein 130 (gp130) ligands from the group of interleukin (IL)-6-like cytokines, wherein the ciliary neurotrophic factor (CNTF) and its analogs are particularly investigated as potentially active therapeutics against adiposity (Febbraio M A, gp130 receptor ligands as potential therapeutic targets for obesity. J. Clin Invest 2007; 117(4):841-9).
CNTF was originally identified as a trophic factor that supports the survival of neurons in the ciliary ganglion of chick embryos (Adler R, Landa K B, Manthorpe M. Varon S. Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons. Science 1979; 204(4400):1434-6). Additional investigations revealed that this effect was not only found with parasympathetic but also with other periphery neurons, such as sympathetic, sensory and motor neurons (Barbin G, Manthorpe M, Varon S. Purification of the chick eye ciliary neuronotrophic factor. J Neurochem 1984; 43(5): 1468-78; Sendtner M, Kreutzberg G W, Thoenen H. Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature 1990; 345(6274):440-1) and also with different central neurons (Ip N Y, Li Y P, van dS, I, Panayotatos N, Alderson R F, Lindsay R M. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J Neurosci 1991; 11(10):3124-34; Hagg T, Quon D, Higaki J, Varon S. Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS. Neuron 1992; 8(1): 145-58; Ip N Y, McClain J, Barrezueta N X, Aldrich T H, Pan L, Li Y, Wiegand S J, Friedman B, Davis S, Yancopoulos G D. The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron 1993; 10(1):89-102; Clatterbuck R E, Price D L, Koliatsos V E. Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system. Proc Natl Acad Sci USA 1993; 90(6):2222-6). CNTF is a molecule that has been strongly conserved during evolution and which functions are mainly restricted to the neuromuscular axis (Perret D, Guillet C, Elson G, Froger J, Plun-Favreau H, Rousseau F, Chabbert M, Gauchert J M, Gascan H. Two different contact sites are recruited by cardiotrophinlike cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines. J Biol Chem 2004; 279(42):43961-70). CNTF shows a number of structural and functional characteristics that distinguish this factor from most other cytokines of the gp130 group. Firstly, it does not possess a signal peptide and consequently cannot be secreted. This led to the early assumption that it is a protein which is stored in the cytosol and released only upon damage of the nerve fibers (Masiakowski P, Liu H X, Radziejewski C, Lottspeich F, Oberthuer W, Wong V, Lindsay R M, Furth M E, Panayotatos N. Recombinant human and rat ciliary neurotrophic factors. J Neurochem 1991; 57(3):1003-12; Stöckli K A, Lottspeich F, Sendtner M, Masiakowski P, Carroll P, Gotz R, Lindholm D, Thoenen H. Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor. Nature 1989; 342(6252):920-3). Further, CNTF in high concentration is able not only to bind to its original alpha receptor (CNTFR), but also to the IL-6 receptor (IL-6R) (Schuster B, Kovaleva M, Sun Y, Regenhard P, Matthews V, Grotzinger J, Rose-John S, Kallen K J. Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF. J Biol Chem 2003; 278(11):9528-35).
Due to its neurotrophic potential, CNTF was tested as a potential therapeutic for different neurodegenerative diseases. Protective effects were shown in animal models (Anderson K D, Panayotatos N, Corcoran T L, Lindsay R M, Wiegand S J. Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease. Proc Natl Acad Sci USA 1996; 93(14):7346-51; Emerich D F, Winn S R, Hantraye P M, Peschanski M, Chen E Y, Chu Y, McDermott P, Baetke E E, Kordower J H. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature 1997; 386(6623):395-9), and a phase I study showed the safety of intracerebrally administered CNTF for Huntington's disease (Bloch J, Bachoud-Levi A C, Deglon N, Lefaucheur J P, Winkel L, Palfi 5, Nguyen J P, Bourdet C, Gaura V, Remy P, Brugieres P, Boisse M F, Baudic S, Cesaro P, Hantraye P, Aebischer P, Peschanski M. Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study. Hum Gene Ther 2004; 15(10):968-75). In contrast, in a clinical phase III study subcutaneously injected CNTF showed no influence on disease progression in patients with amyotrophic lateral sclerosis (Miller R G, Petajan J H, Bryan W W, Armon C, Barohn R J, Goodpasture J C, Hoagland R J, Parry G J, Ross M A, Stromatt S C. A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF) factor in amyotrophic lateral sclerosis. rhCNTF ALS Study Group. Ann Neurol 1996; 39(2):256-60). Some patients in this study developed a dose-dependent weight loss which confirmed the previously observed cachexia after systemic administration of CNTF to mice (Henderson J T, Seniuk N A, Richardson P M, Gauldie J, Roder J C. Systemic administration of ciliary neurotrophic factor induces cachexia in rodents. J Clin Invest 1994; 93(6):2632-8). Accordingly, work was focused on the elucidation of the mechanisms which lead to such a reduction of the body weight to allow the targeted modulation of these mechanisms. It is however to be concluded that CNTF is not a physiological regulator of the weight, and its cachectic effects were observed exclusively after exogenous administration in animal experiments and clinical studies.
Early on it was already known that CNTF from rats has a fourfold higher activity than human CNTF (Masiakowski P, Liu H X, Radziejewski C, Lottspeich F, Oberthuer W, Wong V, Lindsay R M, Furth M E, Panayotatos N. Recombinant human and rat ciliary neurotrophic factors. J Neurochem 1991; 57(3):1003-12). This is due to an amino acid in position 63 of the protein which is arginine (R) in rats and glutamine (Q) in humans. By the substitution Q63R, it was possible to generate a human CNTF variant whose activity corresponds to that of rat CNTF (Panayotatos N, Radziejewski E, Acheson A, Pearsall D, Thadani A, Wong V. Exchange of a single amino acid interconverts the specific activity and gel mobility of human and rat ciliary neurotrophic factors. J Biol Chem 1993; 268(25):19000-3). Further studies showed that also N-terminal or C-terminal deletions to some extent resulted in an increase of activity or at least did not negatively influence CNTF activity (Negro A, Corsa V, Corona G, Grandi C, Skaper S D, Callegaro L. Structure-function studies of human ciliary neurotrophic factor. Neurochem Res 1994; 19(2):223-7; Krüttgen A, Grotzinger J, Kurapkat G, Weis J, Simon R, Thier M, Schroder M, Heinrich P, Wollmer A, Comeau M. Human ciliary neurotrophic factor: a structure function analysis. Biochem J 1995; 309 (Pt 1):215-20). These findings ultimately led to the generation of Axokine®, a CNTF analog which differs from naturally occurring human CNTF by targeted modifications (deletion of the 15 C-terminal amino acids, replacement of cysteine in position 17 by alanine, and replacement of glutamine in position 63 by arginine) (Peterson W M, Wang Q, Tzekova R, Wiegand S J. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci 2000; 20(11):4081-90) and which is tested as a potential therapeutic for adiposity (Preti A. Axokine (Regeneron). IDrugs 2003; 6(7):696-701). Moreover, evidence has been obtained recently that the activity of Axokine® is retained even after simultaneous removal of N- and C-terminal amino acids (Breusing K. Generierung and Charakterisierung eines verbesserten Ciliary Neurotrophic Factors (CNTF). Inauguraldissertation zur Erlangung der Doktorwürde der Medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel 2007).
The CNTF analog Axokine®, developed by the company Regeneron, has already been tested as a potential therapeutic for adiposity. However, in clinical studies the induction of neutralizing antibodies against CNTF has been observed in numerous patients and the substance did not enter the market.
The therapeutic use of CNTF or CNTF analogs, respectively, has been proven problematic so far due to the severe side effects which are associated with their systematic administration. The fact that CNTF in high concentrations is able not only to bind to its original receptor, but also the IL-6R can possibly explain some of the IL-6R-mediated side effects of systemically administered CNTF, such as fever (Shapiro L, Zhang X X, Rupp R G, Wolff S M, Dinarello C A. Ciliary neurotrophic factor is an endogenous pyrogen. Proc Natl Acad Sci USA 1993; 90(18):8614-8) or the induction of an acute phase response in the liver (Dittrich F, Thoenen H, Sendtner M. Ciliary neurotrophic factor: pharmaeokinetics and acute-phase response in rat. Ann Neurol 1994; 35(2):151-63). These effects may be avoided by a monospecifity for the CNTFR.
The induction of neutralizing antibodies against CNTF observed in clinical studies is another so far very problematic aspect when using CNTF as a medicament. These antibodies can both jeopardize the therapeutic effect of the medicament and interfere with the body's own CNTF which is possibly associated with severe consequences. Therefore not only the company Regeneron but also the companies Xencor and Merck have dealt with the mitigation of CNTF immunogenicity (see WO 02/070698 A2; US 2005/0064555 A1 and US 2005/0069987 A1).
Due to the activation of IL-6R CNTF exhibits a B cell and therefore immune stimulating activity. It is hence possible that the pronounced antibody formation that was observed in response to B cell stimulation can be explained by the capability of administered CNTF to bind and activate IL-6R. A CNTF variant which is not able to activate the IL-6R would possibly no longer possess this property. Since on the other hand B cells do not express CNTFR, such a CNTF variant should not be capable of stimulating B cells. It is therefore obvious that the antibody formation against the CNTF variant is prevented in this way.
There is an urgent need for new therapeutics for adiposity and neurological diseases. CNTF variants without affinity or with only low affinity to IL-6R which render it possible to prevent IL-6R-associated side effect are interesting candidates for a new generation of CNTF medicaments for neurological diseases and for the regulation of body weight.